SPECTROPHOTOMETRIC ASSAY FOR ENZYME-MEDIATED UNWINDING OF DOUBLE-STRANDED DNA

被引：36

作者：

HOUSTON, P ^{[1
]}

KODADEK, T ^{[1
]}

机构：

[1] UNIV TEXAS,DEPT CHEM,AUSTIN,TX 78712

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1994年 / 91卷 / 12期

关键词：

HELICASE; FLUORESCENCE; DRUG SCREENING;

D O I：

10.1073/pnas.91.12.5471

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

A method is described for monitoring the enzyme-mediated unwinding of duplex DNA spectrophotometrically. The assay employs a partially duplex oligonucleotide substrate modified at the complementary end with coumarin and fluorescein moieties. When in close proximity the fluorescein quenches the fluorescence of coumarin. However, when the strands are separated by the action of a DNA helicase, the coumarin fluorescence increases greatly. Therefore, the progress of enzyme-mediated DNA unwinding can be measured in real time by fluorescence spectroscopy. This assay provides a simple method to screen for helicase inhibitors, which are of growing interest as potential anticancer agents. The application of this technique to kinetic analyses of the mechanism of action of DNA helicases is also discussed.

引用

页码：5471 / 5474

页数：4

共 18 条

[1]

ARAMATUNGA M, 1993, BIOCHEMISTRY-US, V32, P6815

[2] INVITRO REPLICATION THROUGH NUCLEOSOMES WITHOUT HISTONE DISPLACEMENT [J].